To split human embryonic stem cells grown on Matrigel two matrigel. We start with a confluent six well plate of embryonic stem cells on matrigel, which can be split one to three, to one to five to another matrigel plate. This allows the wells to become confluent again, four to five days after splitting on the day of splitting.
Wash each well that will be split with one XPBS pH 7.4. Then add one milliliter of one milligram per milliliter, dis paste to each well and incubate at 37 degrees Celsius for three minutes. Wash each well three times with one XPBS pH 7.4.
Then add one milliliter of embryonic stem cell media without BF GF and use a cell scraper to scrape the colonies off the bottom of the well. Collect the resuspended embryonic stem cells in a 15 milliliter Falcon tube and pellet by centrifugation at 200 times G for five minutes at room temperature. To reflate the human embryonic stem cells on matrigel plates first, wash the matrigel plates with D-M-E-M-F 12 and add 2.5 milliliters of meth condition Media supplemented with 10 nanograms per mil.
BFGF per well after the cells have been centrifuged reus, suspend the human auric stem cell pellet in an appropriate volume of meth condition. Media supplemented with 10 nanograms per mil. BFGF pipette the cell suspension up and down three times.
Keep in mind that the stem cell clumps from matrigel break up easily. So pipetting three times should be enough to break the clumps, but not make the colonies too small. Finally, aliquot 0.5 milliliters of human amry stem cell suspension per well to achieve three mils per well.
As a final volume, make sure that the clumps of cells are evenly dispersed, and then place the plate in a 37 degree incubator. Depending on the particular amry stem cell line, amry stem cells will become confluent again, four to five days after splitting. When colonies will start to touch each other, it's important to know whether or not your human amry stem cells maintain pluripotency or stemness in culture.
To check pluripotency of the cells during amry stem cell culturing, we use immunofluorescence microscopy to examine the expression of the pluripotency markers, OCT four and SSEA four, one of the wells. Our six well plate is fixed and stained for OC four and SSEA four. If we see that the cells are fluorescently labeled for both of these markers, then we know that our human embryonic stem cells have maintained pluripotency and can be used for our experiments.
We've just shown you how to grow and maintain through potent human embryonic stem cells, amongst embryonic, fibroblasts, feeder cells, and under field free conditions on metro job. So that's it. Thanks for watching and good luck with your experiments.
